The Cytoskeletal Protein Regulation Section has been studying two programs closely linked by an early observation on neurofilament (NF) phosphorylation in mammalian and squid neurons. We demonstrated that cytoskeletal protein phosphorylation in neurons is topographically regulated; i.e., NF tail domain phosphorylation of the many lys-ser-pro (KSP) repeats is restricted to the axonal compartment although kinases, regulators and substrates are also present and active in the perikaryal compartment. An important question is, how is this process normally regulated since deregulation results in neurodegeneration (e.g. Alzheimers Disease, Amyotrophic lateral Sclerosis, Parkinson); i.e., cytoskeletal proteins are aberrantly hyperphosphorylated in neuronal perikarya. Initially we focused on the kinases responsible for the extensive phosphorylation of the numerous lys-ser-pro (KSP) repeats in tail domains of NF proteins and found that the proline directed kinases such as cdk5 and Erk1/2 are primarily involved. In addition, we found that the NF head domains are transiently phosphorylated in the cell body compartment by second messenger protein kinases while axonal phosphorylation of proline-directed ser/thr residues in NF tail domains occurs by tightly regulated proline-directed kinase signal transduction cascades associated with glial / axonal interaction. Aim 1;Topographic regulation of cytoskeletal protein phosphorylation; To further examine this problem, we are studying phosphatases (tyrosine and ser/thr phosphatases), their identification in the mammalian and squid neurons, their localization and regulation, combined with a new lead concerning the role of Pin1, a peptidyl-prolyl isomerase which specifically targets and isomerizes phosphorylated proline-directed ser/thr residues to change function. Aim 2; Role of cdk5 in neuronal survival and neurodegeneration. Cdk5 has been identified as a tightly regulated multifunctional kinase in the nervous system, important in neuronal migration during development, and essential for survival. When cdk5 is deregulated in neuronal stress (oxidative, inflammatory, etc) converts p35 into a smaller activator molecule, p25, which forms a more stable complex with cdk5 (cdk5/p25), abnormally hyperactivates and deregulates cdk5 activity which induces a specific neuronal pathology typically found in such neurodegenerative disorders as Alzheimer, Parkinson and ALS. Mouse models of these diseases can be produced by genetically inducing the expression of high levels of p25 in the brain, thereby implicating hyperactive cdk5 as a major factor in the etiology of these disorders. Currently, most therapeutic approaches targeting the deregulated Cdk5/p25 complex in neurodegenerative disorders have focused primarily on drugs like roscovitine that inhibit cdk5 activity by interfering with the ATP binding domain of the kinase. Most of these drugs, however, lack the specificity required since most cdk kinases (and most kinases) are vulnerable at the ATP binding site. The P5, a 24 amino acid peptide, however, derived from the p35 activator, interacts at a highly specific regulator site. Based on the crystal structure of cdk5/p25 we designed truncated fragments of p25 that interact with the p25/cdk5 interface and inhibit its activity. P5, the peptide exhibiting the most effective inhibition of cdk5/p25 activity, was shown, in a number of in vitro and in vivo (in cells) experimental trials to specifically shut down deregulated cdk5/p25 activity without affecting the activity of the normal cdk5/p35 complex, which is essential for nervous system development and survival. Moreover, the typical neuronal pathology and cell death induced by deregulated cdk5/p25 activity was abrogated by treatment with the P5 peptide. These dramatic results raise the strong possibility that P5 has great therapeutic potential for AD and other neurodegenerative diseases in which deregulated hypperactivated Cdk5 is an etiological factor.